Structure of a central metabolic enzyme determined

Feb 01, 2019

Kiel research team provides key to functional understanding of the human mARC1 enzyme

One of the primary challenges for every living being is to determine the usefulness or harmfulness of ingested substances. In the case of food intake, for example, highly-specialised enzymes are used, which assist with the production of energy from chemically complex food substances. On the other hand, completely different enzymes are involved in breaking down certain non-usable or toxic foreign substances: similar to the immune system, they act as a protective barrier for the body to prevent the absorption of pollutants. In contrast with the specialised digestive enzymes, they are very non-specific, since they need to respond to a wide range of different chemical compounds in order to convert these to excreta. An example of such an enzyme in the human body is the so-called mARC1, which is involved in nitrogen conversion. A Kiel research team described it for the first time around ten years ago, and suspected that it has a special significance for physiology. Now, scientists from the Institute of Pharmacy and the Centre for Biochemistry and Molecular Biology at Kiel University (CAU) have succeeded in producing a high-resolution structural image of the mARC1 enzyme, using a special X-ray crystal structure analysis. This precise depiction of its spatial structure and the molecules contained inside provides the basis for a better functional understanding of the mARC1-controlled metabolic processes. The researchers, who are part of the CAU priority research area "Kiel Life Science" (KLS), recently published their results in the scientific journal Proceedings of the National Academy of Sciences (PNAS).

The Kiel researchers suspected that the enzyme plays a significant role in the metabolism, due to its universal occurrence: it is found not only in every human being, but also in all higher forms of life throughout the animal and plant kingdom. In nitrogen conversion, it triggers biochemical processes that essentially consist of either a reaction or the corresponding reverse reaction - depending on whether it binds or releases oxygen. With these fundamental mechanisms, it can play an important role in the control of pollutants: because nitrogen compounds in some cases produce either particularly toxic or mutagenic degradation products, the enzyme can contribute to their detoxification. At the same time, mARC1 is a special case, since it is only the fourth molybdenum-containing enzyme to be identified in the human metabolism - the xenobiotic metabolism is otherwise mainly characterised by enzymes containing iron.

"We have now been able to look inside the active centre of mARC1 in detail for the first time, and determine how it functions on the basis of its structure," said Professor Axel Scheidig, Director of the Centre for Biochemistry and Molecular Biology (BiMo) at the CAU. "The enzyme can be very effective in reducing pollutants which accumulate in the cell as metabolic products of nitrogen conversion," continued Scheidig. However, depending on the bonds it forms, mARC1 can also work in reverse. Then a toxic effect may occur, due to the conversion caused by the enzyme.

The key to determining the detailed structure was a so-called X-ray crystal structure analysis, which the Kiel research team carried out in cooperation with colleagues from the Deutsches Elektronen-Synchrotron (DESY) in Hamburg. It allowed the very weak signal of the atomic structure itself to be amplified by X-rays, through the interaction of numerous coherently-phased molecules. In this way, the researchers were able to make the structure of the enzyme visible, using the crystal made up of billions of individual molecules. However, for successful crystallisation, they first had to clean the protein molecules of the enzyme and link them with another protein in a lengthy optimisation process, without affecting the functioning of the enzyme while doing so. "We have worked on finding a way to visualise the enzyme structure for about ten years," emphasised Scheidig. "The highly precise depiction of the detailed structure of mARC1 which is now available opens the door to potential exploitation of its functions," he continued.

Now, in further research, the whole spectrum of metabolic processes controlled by mARC1 can be explored, including the organic and inorganic compounds produced. In addition, there is also a second, very similar enzyme, mARC2, whose previously-unknown structure can now also be investigated in detail. The goal of the future work is especially to explore the therapeutic potential of the two closely-related enzymes.

In addition to their importance for nitrogen metabolism, the mARC enzymes are also involved in the conversion of toxic plant substances such as alkaloids, as found in plants like the common ragwort. Here too, it is possible that the chemical reaction produces both harmless and harmful degradation products. Ultimately, the targeted use of enzymes allows the development of novel medicines: for example, the enzymes are involved in the activation of newly-developed blood thinning and anti-cancer drugs. This principle also originated from the working group of Professor Bernd Clement from the Institute of Pharmacy. For future developments, it is conceivable that with the help of mARC, the conversion and thereby the activation of an active substance may be controlled so that it already works in the digestive tract, and does not first have to be absorbed into the bloodstream. Researchers also refer to such medications with delayed activation in the body as "prodrugs". "From a pharmaceutical point of view, by applying this principle, we hope for an increased effectiveness, and potentially reduced side-effects," highlighted Clement.

The genetic profile of mARC1 plays a central role in this further research: here, the Kiel scientists were able to close a knowledge gap, as previous bioinformatic methods were only able to provide an incomplete picture. "We have also identified the genes that underlie the formation of the enzyme in humans," emphasised Clement. "On this basis, we will carry out a systematic functional analysis of the mARC1 enzyme in future, using model organisms," he continued. With the targeted switching on and off of these genes using different experimental methods, comparative statements about the mode of action of the enzyme and the physiological consequences for the organism will be possible.